A novel enzymatic electrochemical biosensor (mCuF/PANI‐nf/HA/Lacc/GCE) was designed for detection of bisphenol A (BPA). The copper ferrite nanoparticles was obtained by co‐precipitation and its surface was modified with ‐NH2 functional organosilane. Polyaniline nanofibers were also synthesized by cyclic voltammetry and characterized by FTIR, XRD, TGA, SEM and TEM, respectively. Then, it was crosslinked with hyaluronic acid as an immobilization matrix for Laccase to adhere to surface of the modified copper ferrites. Cyclic and differential pulse voltammetries were used to evaluate the electrochemical performances of the biosensor, which has a LOD value of 5.40 nM and a LOQ value of 16.20 nM in the 0.01–7.50 μM linear working range. The biosensor was successfully applied for determination of BPA in seawater, canned water and milk samples with recoveries ranging from 96.0 % and 100.7 %. In addition, accuracy of the voltammetric determination method in the real samples was carried out by HPLC and spike/recovery test. The layer‐by‐layer surface modification strategy of the designed mCuF/PANI‐nf/HA/Lacc/GCE biosensor opens a new perspective on both BPA determination and using biopolymer in the structure of enzymatic electrochemical biosensors.